Regulation of important functions including maintenance of homeostasis in the living body, reproduction, development of individuals, metabolism, growth, is control of the nervous, circulatory, immune, digestive or metabolic system, sensory adaptation, and the like, is done by receiving endogenous factors such as various hormones and neurotransmitters or sensory stimulation like light or odor, via specific receptors present on cell membranes, which are furnished in the living body, and responding accordingly. Many of these receptors for hormones or neurotransmitters, which take part in such functional regulation, are coupled to guanine nucleotide-binding proteins (hereinafter, referred to as G proteins), and are characterized by developing a variety of functions through mediation of intracellular signal transduction via activation of the G proteins. In addition, these receptor proteins possess common seven transmembrane regions. Based on the foregoing, these receptors are thus collectively referred to as G protein-coupled receptors or seven transmembrane receptors. As such, it is known that various hormones or neurotransmitters and their receptor proteins are present and interact with each other to play important roles for regulating the biological functions. However, it often remains unclear if there are any other unknown substances (hormones, neurotansmitters, etc.) and receptors to these substances.
In recent years, accumulated sequence information of human genome DNA or various human tissue-derived cDNAs by random sequencing and rapid progress in gene analysis technology have been accelerating the investigation of human genes. With such advance, it has been clarified that there are many genes supposed to encode proteins with unknown functions. G protein-coupled receptors not only have seven transmembrane domains but many common sequences are present in their nucleic acids or amino acids. Thus, these receptors can be precisely identified to be G protein-coupled receptors in such proteins. On the other hand, these G protein-coupled receptor genes are obtained also by polymerase chain reaction (hereinafter abbreviated as PCR) utilizing such a structural similarity. In these G protein-coupled receptors thus obtained so far, ligands to some receptors that are subtypes having high homology in structure to known receptors may be readily predictable but in most cases, their endogenous ligands are unpredictable so that ligands corresponding to these receptors are hardly found. For this reason, these receptors are termed orphan receptors. It is likely that unidentified endogenous ligands to such orphan receptors would participate in biological phenomena poorly analyzed because the ligands were unknown. When such ligands are associated with important physiological effects or pathologic conditions, it is expected that development of these receptor agonists or antagonists will result in breakthrough new drugs (Stadel, J. et al., TiPS, 18, 430-437, 1997; Marchese, A. et al., TiPS, 20, 370-375, 1999; Civelli, O. et al., Brain Res., 848, 63-65, 1999). Until now, however, there are few examples to actually identify ligands to orphan G protein-coupled receptors.
Recently, some groups attempted to investigate ligands to these orphan receptors and reported isolation/structural determination of ligands, which are novel physiologically active peptides. Independently, Reinsheid et al. and Meunier et al. introduced a cDNA coding for orphan G protein-coupled receptor LC132 or ORL1 into animal cells to express a receptor, isolated a novel peptide from porcine brain or rat brain extract, which was named orphanin FQ or nociceptin, with reference to its response and determined its sequence (Reinsheid, R. K. et al., Science, 270, 792-794, 1995; Meunier, J.-C. et al., Nature, 377, 532-535, 1995). This peptide was reported to be associated with pain. Further research on the receptor in knockout mice reveals that the peptide takes part in memory (Manabe, T. et al., Nature, 394, 577-581, 1998).
Subsequently, novel peptides such as PrRP (prolactin releasing peptide), orexin, apelin, ghrelin, GALP (galanin-like peptide) and metastin were isolated as ligands to orphan G protein-coupled receptors by the similar method (Hinuma, S. et al., Nature, 393, 272-276, 1998; Sakurai, T. et al., Cell, 92, 573-585, 1998; Tatemoto, K. et al., Biohem. Biophys. Res. Commun., 251, 471-476, 1998; Kojima, M. et al., Nature, 402, 656-660, 1999; Ohtaki, T. et al., J. Biol. Chem., 274, 37041-37045, 1999; Ohtaki, T. et al., Nature, 411, 613-617, 2001). So far, among them, it has been reported that orexin relates to feeding and sleeping (Sakurai, T. et al., Cell, 92, 573-585, 1998; Lin, L. et al., Cell, 98, 365-376, 1999; Chemelli, R. M. et al., Cell, 98, 437-451, 1999), and that ghrelin exhibits feeding accentuation activity (Tschop, M. et al., Nature, 407, 908-913, 2000; Nakazato, M. et al., Nature, 409, 194-198, 2001). Further, it has been suggested that metastin has a possibility to suppress metastasis (Ohtaki, T. et al., Nature, 411, 613-617, 2001).
On the other hand, some receptors to physiologically active peptides, which were so far unknown, were clarified according to the similar manner. It was revealed that a receptor to motilin associated with contraction of intestinal tracts was GPR38 (Feighner, S. D. et al., Science, 284, 2184-2188, 1999). Furthermore, SLC-1 (MCHR1) (Chambers, J. et al., Nature, 400, 261-265, 1999; Saito, Y. et al., Nature, 400, 265-269, 1999; Shimomura, Y. et al., Biochem. Biophys. Res. Commun., 261, 622-626, 1999; Lembo, P. M. C. et al., Nature Cell Biol., 1, 267-271, 1999; Bachner, D. et al., FEBS Lett., 457, 522-524, 1999) and SLT (MCHR2) (Mori, M. et al., Biochem. Biophys. Res. Commun., 283, 1013-1018, 2001; Hill, J. et al., J. Biol. Chem., 276, 20125-20129, 2001; Sailer, A. W. et al., Proc. Natl. Acad. Sci. USA, 98, 7564-7569, 2001; An, S. et al., Proc. Natl. Acad. Sci. USA, 98, 7576-7581, 2001) were identified as a receptor to MCH. Also, it was reported that GPR14 (SENR) was a receptor to urotensin II (Ames, R. S. et al., Nature, 401, 282-286, 1999; Mori, M. et al., Biochem. Biophys. Res. Commun., 265, 123-129, 1999; Nothacker, H.-P. et al., Nature Cell Biol., 1, 383-385, 1999, Liu, Q. et al., Biochem. Biophys. Res. Commun., 266, 174-178, 1999) and that FM3 (GPR66) and FM4 were a receptor to neuromedin U (Howard, A. D. et al., Nature, 406, 70-74, 2000; Szekeres, P. G. et al., J. Biol. Chem., 275, 20274-20250, 2000; Fujii, R. et al., J. Biol. Chem., 275, 221068-21074, 2000; Hosoya, M. et al., J. Biol. Chem., 275, 29528-29532, 2000; Raddatz, R. et al., J. Biol. Chem., 275, 39482-39486, 2000; Kojima, M. et al., Biochem. Biophys. Res. Commun., 276, 435-438, 2000). It was shown that MCH took part in obesity since its knockout mouse showed the reduced body weight and lean phenotype (Shimada, M. et al., Nature, 396, 670-674, 1998), and because its receptor was identified, it became possible to explore a receptor antagonist likely to be an anti-obesity agent. It is further reported that urotensin II shows a potent action on the cardiocirculatory system, since it induces heart ischemia by intravenous injection to monkey (Ames, R. S. et al., Nature, 401, 282-286, 1999). Neuromedin U was shown to function as suppression of feeding (Howard, A. D. et al., Nature, 406, 70-74, 2000; Kojima, M. et al., Biochem. Biophys. Res. Commun., 276, 435-438, 2000).
As described above, orphan receptors and ligands thereto often take part in a new physiological activity, and it is expected that their identification will lead to development of new drugs. However, it is known that research on ligands to orphan receptors is accompanied by many difficulties. For example, since it is generally unknown that orphan receptor, which is expressed in the cells, responds to the ligand and thereafter second signal transduction is activated, it is necessary for investigation of various response system. Further, since it is not easily predicted where the tissues having ligand are, various tissue extracts should be prepared. Furthermore, when the ligand is a peptide, since the ligand level necessary for stimulation of the receptor is enough to be extremely low concentration, there is many cases that in vivo level of such a ligand is extremely trace. In addition, since an activity of the peptide is lost due to digestion by protease and recovery of the peptide during purification steps is retarded due to non-specific adsorption, the fact that an amount of the peptide to be needed for determination of the structure is extracted from the living body and isolated is generally very difficult. Due to these issues, the presence of many orphan receptors was unraveled, but due to the foregoing problems, only a very small part of ligands to these receptors were discovered so far.
By finding out a ligand of novel G protein-coupled receptor and directly applying the ligand, or by using a screening system for medicine with the ligand, it is desired to develop a medicine having a quite novel mechanism of action.